PROJECTSUMMARY Growing evidence suggest that central nervous system (CNS) inflammation is a major pathophysiological processinParkinson?sdisease(PD).Theinflammationischaracterizedbyanincreaseinthenumberofmicroglia and in the levels of proinflammatory cytokines and chemokines. The proinflammatory cytokines, which are released when microglia are activated, could exacerbate ?-synuclein (?-syn) aggregation and neuronal death ultimatelydrivingthechronicprogressionofPD.Dyingordeaddopaminergicneuronsreleased?-synaggregates canbetakenupintothemicroglia,whichleadstomicrogliaactivation.Thisinturncontributestotheaccelerated progressionofneurodegenerativeprocesses.Therefore,identificationofmoleculartargetsinmicrogliathatcan bemodulatedtoinhibittheirinflammatoryresponsemayprovidenewstrategyforthetreatmentofPD.However, there is a lack of knowledge about the cellular mechanisms underlying microglia-mediated neuroinflammatory events following stimulation with ?-syn aggregates. To gain a comprehensive understanding of the cellular mechanisms of ?-syn aggregates induced microglial activation, we conducted RNAseq analysis using ?-syn preformedfibrils(?-synPFF)activatedmicroglia.Fromthescreening,weidentifiedNOD2/RIPK2complexasa keymodulatorinneuroinflammatoryresponsesdueto?-synaggregate.Inourpreliminarystudies,wefoundthat ?-synaggregatesactivateNOD2/RIPK2mediatedinflammatoryresponsesinmicrogliatoexacerbatesneuronal deaththroughaseriesofsignalingevents,whichincludedM1microglialactivationandtoxicA1typeastrocytes conversionbysecretingIL-1?,TNF?andC1q.Ontheotherhand,wefoundthatthedepletionofNOD2/RIPK2 complex protects neuronal death via blocking microglia-mediated A1 astrocyte conversion in vitro and in vivo. Tofurtherconfirmandexpandournovelpreliminaryfindings,wewillsystematicallypursuethefollowingspecific aims:(i)todefinethelevelsofNOD2,RIPK2mRNAsandproteinsandthelevelsofphosphorylationofRIPK2 inmicrogliainmousemodelsof?-syninduceddegenerationandPDpostmortembrains,ii)tocharacterizethe interactionbetweenNOD2and?-synaggregatesinmicrogliaanddefinehow?-synPFFbindingtoNOD2affects NOD2/RIPK2 dependent signaling pathways including RIPK2 activation/ubiquitination and MAPKs and NF-kB activation in microglia, iii) determine the depletion effect of NOD2/RIPK2 signaling in microglia-induced A1 astrocytesformationdueto?-synaggregatesbothinvitroandinvivo,and(iv)todeterminetheeffectsofblocking ofNOD2/RIPK2signalingin?-synPFFinducedmodelofPDthroughgeneticablationofNOD2andRIPK2and pharmacologicalinhibitionofRIPK2.Cellular,neurochemical,neuropathologicalandbehavioralapproachesin vitro and in vivo will be used to achieve these specific aims. Taken together, understanding the role of NOD2/RIPK2 dependent signaling in ?-syn aggregates-induced microglial activation, A1 astrocyte conversion andDAneuronaldeathwillnotonlyprovidenovelmechanisticinsightsintotheprogressionofneurodegenerative processesinPD,butalsoultimatelyleadtothedevelopmentofnewtherapeuticstrategiesforPD.